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Biomedical subjects

M Barthélémy

Publications and source records attributed to M Barthélémy.

At least 19 recordsLinked to original sources

Plasmodium chabaudi chabaudi chronic malaria and pathologies of the urogenital tract in male and female BALB/c mice.

BALB/c mice of both sexes were infected with a non-virulent strain of Plasmodium chabaudi chabaudi and any pathologies occurring in the urogenital tract and its accessory glands were investigated. Organs and tracts were removed from infected and control mice at 15, 40 and 100 days post-injection, weighed and processed for macroscopical and histological analyses. The relative weights of preputial, clitoral glands and testes were modified in infected mice during the 40 days following infection. The preputial glands show a marked hypotrophy at 15 days post-infection. The bladders of half of the infected female mice and a few infected male mice displayed a conspicuous haemoglobinuria and frequent interstitial cystitis that worsened throughout the experiment. Also, several chronic inflammatory reactions were detected in the prostates, preputial and clitoral glands up to 100 days post-infection. A probable cause of such a divergence in the characteristics of the infection and in the nature of the pathologies identified in infected male and female mice is the interaction between the infection and the immune and endocrine systems of the host. The cause of the pathologies and their consequences on the host condition are discussed.

Animals↗

Solution structure of microcin J25, the single macrocyclic antimicrobial peptide from Escherichia coli.

The three-dimensional solution structure of microcin J25, the single cyclic representative of the microcin antimicrobial peptide class produced by enteric bacteria, was determined using two-dimensional 1H NMR spectroscopy and molecular modeling. This hydrophobic 21-residue peptide exhibits potent activity directed to Gram-negative bacteria. Its primary structure, cyclo(-V1GIGTPISFY10GGGAGHVPEY20F-), has been determined previously [Blond, A., Péduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthélémy, M., Prigent, Y., Salomón, R.A., Farías, R.N., Moreno, F. & Rebuffat, S. (1999) Eur. J. Biochem., 259, 747-755]. Conformational parameters (3JNHCalphaH coupling constants, quantitative nuclear Overhauser enhancement data, chemical shift deviations, temperature coefficients of amide protons, NH-ND exchange rates) were obtained in methanol solution. Structural restraints consisting of 190 interproton distances inferred from NOE data, 11 phi backbone dihedral angle and 9 chi1 angle restraints derived from the coupling constants and three hydrogen bonds in agreement with the amide exchange rates were used as input for simulated annealing calculations and energy minimization in the program XPLOR. Microcin J25 adopts a well-defined compact structure consisting of a distorted antiparallel beta sheet, which is twisted and folded back on itself, thus resulting in three loops. Residues 7-10 and 17-20 form the more regular part of the beta sheet. The region encompassing residues Gly11-His16 consists of a distorted beta hairpin, which divides into two small loops and is stabilized by an inverse gamma turn and a type I' beta turn. The reversal of the chain leading to the Phe21-Pro6 loop results from a mixed beta/gamma turn. A cavity, in which the hydrophilic Ser8 side-chain is confined, is delimited by two crab pincer-like regions that comprise residues 6-8 and 18-1.

Amino Acid Sequence↗

The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli.

Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non-SOS-dependent pathway [R. A. Salomon and R. Farias (1992) J. Bacteriol. 174, 7428-7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin-overproducing Escherichia coli strain by reverse-phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two-dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21-residue peptide, cyclo(-Val1-Gly-Ile-Gly-Thr- Pro-Ile-Ser-Phe-Tyr-Gly-Gly-Gly-Ala-Gly-His-Val-Pro-Glu-Tyr-Phe21- ). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe21-Val1 bond and led to a single peptide, MccJ25-L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 microgram.mL-1), the thermolysin-linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N-terminal extremity, the present study shows that removal of this extension and head-tail cyclization of the resulting propeptide are the only post-translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.

Amino Acid Sequence↗

Scaling for the critical percolation backbone.

We study the backbone connecting two given sites of a two-dimensional lattice separated by an arbitrary distance r in a system of size L at the percolation threshold. We find a scaling form for the average backbone mass: approximately L(dB)G(r/L), where G can be well approximated by a power law for 0 approximately L(dB-psi)r(psi) for the entire range 0<r<L. We also propose a scaling form for the probability distribution P(M(B)) of backbone mass for a given r. For r approximately L, P(MB) is peaked around L(dB), whereas for r<<L, P(M(B)) decreases as a power law, M(-tauB)B, with tauB approximately 1.20+/-0.03. The exponents psi and tauB satisfy the relation psi=dB(tauB-1), and psi is the codimension of the backbone, psi=d-dB.

Journal Article↗

Clinical inhibitor-resistant mutants of the beta-lactamase TEM-1 at amino-acid position 69. Kinetic analysis and molecular modelling.

The kinetic parameters of three IRT (Inhibitor-Resistant-TEM-derived-) beta-lactamases (IRT-5, IRT-6 and IRT-I69) were determined for substrates and the beta-lactamase inhibitors: clavulanic acid, sulbactam and tazobactam, and compared with those of TEM-1 beta-lactamase. The catalytic behaviour of the beta-lactamases towards substrates and inhibitors was correlated with the properties of the amino acid at position ABL69. The three IRT beta-lactamases contain at that position a residue Ile, Leu and Val, amino acids whose side-chain are branched. Molecular modelling shows that the methyl groups of Ile-69 (C gamma 2) and Val-69 (C gamma 1) produced steric constraints with the side chain of Asn-170 as well as the main chain nitrogen of Ser-70, a residue contributing to the oxyanion hole. We suggest that hydrophobicity could be the main factor responsible for the kinetic properties of Met69Leu (IRT-5), as no steric effects could be detected by molecular modelling. Hydrophobicity and steric constraints are combined in Met69Ile and Met69Val, IRT-I69 and IRT-6, respectively.

Anti-Bacterial Agents↗

Phenotypic study of resistance of beta-lactamase-inhibitor-resistant TEM enzymes which differ by naturally occurring variations and by site-directed substitution at Asp276.

At this time an amino acid substitution at position 276 in the TEM-1 enzyme is associated with an additional substitution at position 69 in natural beta-lactamase-inhibitor-resistant (IRT) beta-lactamases. The effect of the Asn276-->Asp substitution on resistance was assessed with the Asn276Asp variant, generated by site-directed mutagenesis. The mutant was resistant to beta-lactamase inhibitors, but the MICs of amoxicillin combined with clavulanic acid or tazobactam were strikingly different for E. coli strains producing the Asn276Asp variant and those producing naturally occurring IRTs with single or double substitutions. The inhibitory effects of clavulanic acid and tazobactam were the same in IRTs with substitutions at position 69 (IRT-5 and IRT-6). The effect of clavulanic acid on the MICs of amoxicillin for the Asn276Asp variant was greater than that of tazobactam. In IRTs with double substitutions, at positions 69 plus 276 (IRT-4, IRT-7, and IRT-8) or 69 plus 275 (IRT-14), tazobactam was a more potent inhibitor than clavulanic acid. The effect of the Asn276-->Asp substitution on the values of the kinetic constants and the concentration required to inhibit by 50% the hydrolysis of benzylpenicillin confirms that this single mutation is responsible for resistance to beta-lactamase inhibitors. Molecular modeling of the Asn276Asp mutant shows that Asp276 can form two salt bonds with Arg244 close to the penicillin-binding cavity. The addition of the Asp276 mutation to that preexisting at position 69 confers a higher selective advantage to bacteria, as shown by the reduction in beta-lactamase inhibitor efficiencies of the double variants. Therefore, the emergence of multiple mutations in TEM beta-lactamases by virtue of the use of beta-lactamase inhibitors increases selection pressure resulting in the convergent evolution of resistant strains.

Amoxicillin↗

Characterization and amino acid sequence analysis of a new oxyimino cephalosporin-hydrolyzing class A beta-lactamase from Serratia fonticola CUV.

Serratia fonticola CUV produces two isoenzymes (forms I and II) with beta-lactamase activity which were purified by a five-step procedure. The isoenzymes had identical kinetic parameters and isoelectric point (pI = 8.12). They were characterized by a specific activity towards benzylpenicillin of 1650 U/mg. The beta-lactamase hydrolyzed benzylpenicillin, amoxycillin, ureidopenicillins, first- and second-generation cephalosporins. Carboxypenicillins and isoxazolylpenicillins were hydrolyzed to a lesser extent. Towards cefotaxime and ceftriaxone (third-generation cephalosporins), the S. fonticola enzyme exhibited catalytic efficiencies much higher than those of MEN-1 and extended-spectrum TEM derivative beta-lactamases. The beta-lactamase from S. fonticola was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. The purified isoenzymes were digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino acid sequence determinations of the resulting peptides allowed the alignment of 267 amino acid residues (Swiss-Prot, accession number P 80545) for form I beta-lactamase. Form II is five residues shorter than form I at its N-terminus. From amino acid sequence comparisons, S. fonticola CUV beta-lactamase was found to share more than 69.3% identity with the chromosomally encoded beta-lactamases of Klebsiella oxytoca, Proteus vulgaris, Citrobacter diversus and the plasmid-mediated enzymes MEN-1 and Toho-1. Therefore, the oxyimino cephalosporin-hydrolyzing beta-lactamase of S. fonticola belongs to Ambler's class A. Contribution of the serine at ABL 237 in the broad-spectrum activity of these beta-lactamases is discussed.

Amino Acid Sequence↗

Oxacillin-hydrolyzing beta-lactamase involved in resistance to imipenem in Acinetobacter baumannii.

Acinetobacter baumannii strain A148, a clinical isolate resistant to imipenem (MIC = 32 mg l-1), synthesized two beta-lactamases with pIs 6.3 and > 9.2. The pI 6.3 enzyme hydrolyzed the penicillins, including isoxazoylpenicillins, first-, second- and, to a lesser extent, third-generation cephalosporins. It was inhibited by chloride ions and by the penem beta-lactamase inhibitor BRL 42715. Clavulanate was a weak inhibitor and EDTA did not affect the beta-lactamase activity. This enzyme also hydrolyzed imipenem with a catalytic efficiency (Kcat/Km) of 1500 mM-1 s-1. Moreover, this purified beta-lactamase produced a positive microbiological clover-leaf test with imipenem. Therefore, the pI 6.3 beta-lactamase was considered to be involved in the imipenem resistance of A. baumannii strain A148.

Acinetobacter↗

Rotational coronary atherectomy with adjunctive balloon angioplasty: evaluation of lumen enlargement by quantitative angiographic analysis.

To evaluate the mechanisms of lumen enlargement and the respective contributions of rotational coronary atherectomy (RA) and adjunctive percutaneous transluminal coronary balloon angioplasty (PTCA), serial measurements were recorded in 70 consecutive patients by quantitative coronary angiography before RA, after RA, after adjunctive PTCA, and 24 hours later. Minimal luminal diameter (MLD) increased from 0.85 +/- 0.31 mm to 1.42 +/- 0.27 mm (p < 0.001) after RA and to 2.20 +/- 0.46 mm (p < 0.001) after PTCA. Minimal luminal area (MLA) increased from 0.64 +/- 0.50 mm2 to 1.63 +/- 0.60 mm2 (p < 0.001) after RA and to 3.97 +/- 1.68 mm2 (p < 0.001) after PTCA. Both 24-hour MLD and MLA showed a trend toward reduced values (2.07 +/- 0.45 mm and 3.52 +/- 1.70 mm2, respectively) when compared with immediate results after PTCA. The absolute gains in MLD after RA and after PTCA were 0.56 +/- 0.24 mm and 0.79 +/- 0.38 mm, respectively (p < 0.01). The absolute gains in MLA after RA and after PTCA were 0.99 +/- 0.49 mm2 and 2.34 +/- 1.41 mm2, respectively (p < 0.001). The respective contributions of RA and PTCA are highly variable, but in general, balloon dilatation accounts for most of the gain in lumen area and therefore is not an adjunctive but a primary technique.

Aged↗

Characterization and amino acid sequence of the OXY-2 group beta-lactamase of pI 5.7 isolated from aztreonam-resistant Klebsiella oxytoca strain HB60.

Klebsiella oxytoca strain HB60 is highly resistant to cefoperazone and aztreonam (MICs = 128 mg/L). It produces a chromosomally encoded beta-lactamase of pI 5.7 which was highly efficient against penicillins, first-generation cephalosporins and cefoperazone, a non-oxyimino third-generation cephalosporin. Aztreonam and oxyimino broad-spectrum cephalosporins were less good substrates. The beta-lactamase activity was susceptible to inhibition by clavulanic acid (IC50 = 1 microM). The enzyme purified to homogeneity had a specific activity towards benzylpenicillin of 3670 U/mg. The 263 amino acid residues of the protein were sequenced by Edman degradation of proteolytic peptides. The beta-lactamase was shown to belong to the OXY-2 group as it had only one amino acid substitution (Asn for Asp at ABL position 197) compared with the beta-lactamase (pI 5.2) from the aztreonam-susceptible K. oxytoca strain SL911 and two substitutions (Ala223 for Val and Asp255 for Asn) compared with the beta-lactamase (pI 6.4) from the aztreonam-resistant K. oxytoca strain D488. These three OXY-2-group enzymes behave in the same way towards beta-lactam antibiotics. The variability in the resistance of these K. oxytoca strains would thus seem to be due to variation in the level of production of the beta-lactamases rather than to structural alteration of the enzymes.

Amino Acid Sequence↗

Properties of IRT-14 (TEM-45), a newly characterized mutant of TEM-type beta-lactamases.

IRT-14 (TEM-45) is a new mutant TEM-type beta-lactamase that was isolated from clinical Escherichia coli P37 and that confers resistance to broad-spectrum penicillins with reduced sensitivity to beta-lactamase inhibitors. The MICs of amoxicillin alone and of amoxicillin combined with 2 micrograms of clavulanic acid or 2 micrograms of tazobactam per ml were 4,096, 2,048, and 1,024 micrograms/ml, respectively. The strain was susceptible to cephalosporins, aztreonam, moxalactam, and imipenem. The enzyme was purified to homogeneity, and values of the kinetic parameters Kcat, Km, and Kcat/Km were determined for different substrates. This enzyme, with a pI of 5.2, was found to have reduced affinity for broad-spectrum penicillins and cephalosporins. The values of 50% inhibitory concentrations of clavulanic acid, sulbactam, tazobactam, and brobactam are correlated with the higher KmS for substrates. The resistance of E. coli P37 to mechanism-based inactivators results from a higher level of production of the TEM-derived enzyme due to the G-to-T substitution at position 162 (G-162-->T) in the promoter region of blaTEM and from the structural modifications resulting from the Met-69-->Leu and Arg-275-->Gln substitutions that characterize IRT-14 beta-lactamase.

Anti-Bacterial Agents↗

An additional ionic bond suggested by molecular modelling of TEM-2 might induce a slight discrepancy between catalytic properties of TEM-1 and TEM-2 beta-lactamases.

The plasmid-mediated TEM-1 and TEM-2 beta-lactamases are the most commonly encountered among Gram-negative bacteria. They belong to molecular class A, and differ by one amino acid at position 39:TEM-1 have a glutamine and TEM-2 a lysine. Kinetic parameters (kcat and Km) and catalytic efficiency (kcat/Km) of TEM-1 and TEM-2 beta-lactamases are slightly, but significantly different. For all antibiotics except methicillin and cefazolin, the catalytic efficiency values of TEM-2 are clearly greater than that of TEM-1. Molecular modelling of TEM-2, when compared to that of TEM-1, showed an additional ionic bond between Lys-39 and Glu-281.

Catalysis↗

Characterization of a novel elastase inhibitor from a fan coral.

An acidic hydromethanolic extract of the tropical gorgonian Melithea cf. stormii exhibited anti-elastase activity. From the polypeptidic mixture we isolated and purified to homogeneity a protein with a molecular mass determined at 21,159 Da by Maldi/Tof mass spectrometric analysis. The novel protein of marine invertebrate origin strongly inhibited amidolysis of Suc(Ala) 3pNA by porcine pancreatic elastase (PPE) and was labelled iela melst. The N-terminal aminoacid sequence of its 39-first residues revealed the characteristics of a non-classical Kazal-type domain. Iela melst behaved as a reversible tight-binding inhibitor of PPE. The competitive inhibition followed Cha's mechanism A with an equilibrium dissociation constant, Ki, calculated as 1.5 x 10(-9) M.

Amino Acid Sequence↗

[Identification of a cephalosporinase susceptible to clavulanic acid in a clinical strain of Serratia fonticola].

We analyzed the beta-lactamase production of a Serratia fonticola isolated for its resistance to cefuroxime (Minimum Inhibitory Concentration > 256 mg/l) in December 1993 from a patient hospitalized in Meaux. The wild strain was resistant to amoxycillin but sensitive to augmentin, that suggested the production of a beta-lactamase susceptible to clavulanic acid. For the wild strain, beta-lactamase production was inducible and only one enzyme with an isoelectric point of 8.12 was detected. beta-lactamase production was 16 mU/mg for non-induced extracts and ranged from 100 to 230 mU/mg in the presence of inducing beta-lactams (enzyme activity was measured with penicillin G as substrate). On a Szybalski gradient a constitutive strain was obtained. Its enzyme production was 13,000 mU/mg. The kinetics and isoelectric points of the enzymes produced by the two strains were identical. This beta-lactamase hydrolyzes penicillins (amoxycillin: Vm = 60 relative to penicillin G = 100, ticarcillin: 15), first generation cephalosporins (cephalothin Vm = 930). However, this enzyme hydrolyzes efficiently oxyimino-cephalosporins: cefuroxime (Vm = 70) and cefotaxime (Vm = 120), but cephamycins are not substrates. Clavulanic acid has a very good affinity for this beta-lactamase (Ki = 0.09 microM) which is inactivated progressively (I50 = 0.045 microgram/ml). These properties shows some similarities with those of the class A beta-lactamases of P. vulgaris RO104 (pI = 8.3), P. penneri 14HBC (pI = 6.65) and the plasmid-mediated extended-spectrum MEN-1 (pI = 8.4).

Aged↗

[Comparative study of the efficacy and tolerability of 2 modalities of intravenous cibenzoline administration in the reduction of recent spontaneous atrial arrhythmia].

This multicentre, single blind, parallel group study compared the efficacy and clinical and electrocardiographic tolerance of a 2 minute intravenous administration of cibenzoline at a dose of 1.2 mg.kg-1 with that of a 10 minute 1.75 mg.kg-1 infusion in patients presenting with spontaneous atrial fibrillation (AF) for less than 6 weeks. Sixty-two patients (40 men and 22 women) with an average age of 62 years and presenting with sustained AF for at least 30 minutes with a ventricular rate greater than or equal to 80 bpm were randomly assigned to groups and received via the intravenous route either one of the two treatments. Efficacy (return to sinus rhythm) was assessed by an ECG recording every 5 minutes and at 45 and 60 minutes thereafter. Sixty-one of the 62 randomised patients were assessed for efficacy. Cibenzoline, administered in the form of a bolus or infusion, proved effective within one hour in 4 patients in each group (13%) and arrhythmia persisted with ventricular rate of less than 80 bpm in 10 (33%) and 5 (16%) of the patients respectively. In patients in whom sinus rhythm was not restored, ventricular rate was significantly reduced by cibenzoline. The patients in whom normal rhythm was restored under one of these treatment regimens were significantly younger. Patients in whom rhythm returned to normal following the administration of the bolus had AF of significantly more recent onset than that of the patients in whom abnormal rhythm persisted, whilst the history of the AF did not differ significantly between these two types of response after the infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Arrhythmia Agents↗

Chromosomally encoded cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A.

Proteus vulgaris RO104 strain produces a chromosomally encoded beta-lactamase that confers resistance to various beta-lactam antibiotics including methoxyimino third-generation cephalosporins. The beta-lactamase hydrolyzes first- and second-generation cephalosporins efficiently and cefotaxime to a lesser extent. Catalytic activity is inhibited by low concentrations of clavulanic acid and sulbactam. By its broad-spectrum substrate profile, beta-lactamase of Proteus vulgaris RO104 belongs to the group 2e defined by Bush. The protein purified to homogeneity by a four-step procedure was characterized by a pI of 8.31 and a specific activity of 1200 U/mg. The beta-lactamase was digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino-acid sequence determinations of the resulting peptides allowed the alignment of the 271 amino-acid residues of the protein which did not contain any cysteine residue. From amino-acid sequence comparisons, Proteus vulgaris RO104 beta-lactamase was found to share about 68% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca D488 and E23004. Therefore, the cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A.

Amino Acid Sequence↗

Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to beta-lactamase inhibitors.

The clinical isolate Escherichia coli PEY was highly resistant to amoxycillin, ticarcillin and piperacillin associated to beta-lactamase inhibitors such as clavulanic acid, sulbactam, tazobactam and brobactam but susceptible to cephalosporins, aztreonam and imipenem. The susceptibility to mecillinam indicated that this phenotype was not related to hyperproduction of the TEM-1 beta-lactamase. E. coli PEY produced a new plasmid-mediated inhibitor-resistant beta-lactamase of pI 5.2, which was named IRT-4. The determination of the amino acid sequence (Swiss-Prot accession number, P00810) of the purified protein indicated that IRT-4 differed from TEM-1 by two substitutions: Leu for Met-69 (ABL numbering) and Asp for Asn-276. A Met-69-Leu variant of TEM-1, obtained by site-directed mutagenesis, has been described as resistant to clavulanate. The Asp for Asn-276 substitution has not been reported previously. The side chains of Asp-276 and Arg-244 were expected to interact. Determinations of 50% inhibitory concentrations of beta-lactamase inhibitors and substrate profile of IRT-4 suggested that such an ionic bond was implicated in the alteration of the mechanistic process of TEM-1 beta-lactamase.

Amino Acid Sequence↗

Val-237 for Ala substitution in the TEM-2 beta-lactamase dramatically alters the catalytic efficiencies towards carbenicillin and ticarcillin.

The mutant 554 of TEM-2 beta-lactamase was selected for a decrease in the resistance to carbenicillin of an Escherichia coli K12 carrier. The amino acid sequence of the mutant beta-lactamase was determined by manual Edman degradation analysis of proteolytic peptides. A single substitution Val for Ala was localized at position 237. The mutant exhibited only 2% of the catalytic efficiency of the wild-type enzyme towards carbenicillin and ticarcillin, whereas it retained 30-60% of the hydrolytic activity towards other penicillin and cephalosporin substrates. Carfecillin, the phenyl ester of the side-chain carboxyl group of carbenicillin, was hydrolysed as a good substrate. This suggests that the behaviour of the mutant enzyme towards carbenicillin may result from ionic rather than steric constraints. A molecular model of the Val-237 TEM-2 mutant suggests possible electrostatic interaction between Glu-171 and the carboxylic group of the side chain of carbenicillin.

Amino Acid Sequence↗